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Flexible Tools for Gene Expression and Silencingin Tomato1[W][OA]

Ana I. Fernandez2, Nicolas Viron2, Moftah Alhagdow, Mansour Karimi, Matthew Jones, Ziva Amsellem,Adrien Sicard, Anna Czerednik, Gerco Angenent, Donald Grierson, Sean May, Graham Seymour,Yuval Eshed, Martine Lemaire-Chamley, Christophe Rothan3, and Pierre Hilson3*

Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Ghent, Belgium (A.I.F.,M.K., P.H.); Department of Plant Biotechnology and Genetics, Ghent University, 9052 Ghent, Belgium (A.I.F.,M.K., P.H.); Institut National de la Recherche Agronomique, Unite Mixte de Recherche 619, CentreBordeaux-Aquitaine, 33883 Villenave d’Ornon, France (N.V., M.A., A.S., M.L.-C., C.R.); Plant and CropScience Division, University of Nottingham, Loughborough, LE12 5RD, United Kingdom (M.J., D.G., S.M., G.S.);Department of Plant Sciences, Weizmann Institute of Science, 76100 Rehovot, Israel (Z.A., Y.E.); RadboudUniversiteit Nijmegen, 6525 HP Nijmegen, The Netherlands (A.C.); and Plant Research International,6700 Wageningen, The Netherlands (G.A.)

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation andtransformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate theassembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available genemanipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruitdevelopment, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entryclones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC fromArabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localizedGUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and infruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcriptionof artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes onlyinvolving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene wasdemonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specificArabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression.Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated toSolanaceae resources.

Solanaceae provide the world’s most importantvegetable crops, including tomato (Solanum lycopersi-cum), potato (Solanum tuberosum), and pepper (Capsi-cum annuum), and are a model eukaryote family forevolutionary, genetic, and genomic studies. More than3,000 Solanaceous species grow in habitats rangingfrom rain forests to deserts and mountains with reg-ular snowfall. Thanks to conserved genome organiza-

tion, this family is an excellent subject to explore thegenetic and molecular basis of adaptation to diverseenvironments. Within the Solanaceae, tomato is abroadly used model system for studying plant-microbe interactions and the biology of fleshy fruits,owing to its simple diploid genetics, the short gener-ation time, the routine protocols for production oftransgenic plants, and its exceptional publicly avail-able genetic and genomic resources. These resourcesinclude large collections of single-gene mutations, anextensive EST database, a high-density genetic map,microarrays, an emerging genome sequence, and well-characterized populations designed for genetic map-ping of simple and quantitative trait loci, and they areused by an active Solanaceae research communityinvolving research laboratories in more than 30 coun-tries focusing on both basic and strategic research(Knapp, 2002; Mueller et al., 2005; Wu et al., 2006).

The first draft sequence of the entire euchromaticportion of the tomato genome will soon be available(Mueller et al., 2009). As already witnessed for otherresearch communities dedicated to key species, the

1 This work is supported by the European Solanaceae Integratedproject, EU-SOL, through the 6th Framework Programme of theEuropean Commission (FOOD–CT–2006–016214).

2 These authors contributed equally to the article.3 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Pierre Hilson ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.109.147546

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availability of a well-annotated genome will mark theonset of new lines of investigations and will greatlyfacilitate the characterization of genetic functions.Taking advantage of the EU-SOL European integratedproject (http://www.eu-sol.net/), we initiated a col-laborative effort for the construction of publicly avail-able tools enabling gene manipulation at specificstages of tomato fruit development and in well-defined tissues and cell types. Tomato is a berry witha thick pericarp that encloses many seeds. The fruitresults from the fusion of a number of carpels, theovary wall becoming the fleshy pericarp. Pollinationtriggers cell proliferation, followed by extensive cellexpansion, resulting in a mature fruit that ranges from1 to 1,000 g (Gillaspy et al., 1993; Lemaire-Chamleyet al., 2005; Gonzalez et al., 2007). Fruit developmentand ripening are also characterized by dramatic met-abolic shifts, including the conversion of starch tosugars, chloroplasts to chromoplasts, and extensivecell wall disassembly, all under tight genetic control(Giovannoni, 2004). Changes in the spatial or temporalexpression of regulatory genes or downstream effectorscontrolling these processes will substantially affect fruittissue properties and the ripening process (Manninget al., 2006; Giovannoni, 2007). Indeed, the ability toalter subtly the timing and location of expression of keyregulators is an important goal for crop biotechnologybut is often constrained by lack of suitable promoters.

To this end, a core set of versatile resources designedfor temporal and tissue-specific manipulation of geneexpression in the tomato fruit has been created byindependent laboratories. All clones and vectors wereconstructed with the flexible Gateway recombina-tional cloning technology (for review, see Karimiet al., 2007a). Promoters that displayed tissue-specifictranscriptional activity have been captured as Gate-

way entry clones. These regulatory sequences can becombined at will with any gene, hairpin RNA(hpRNA), expression cassette, or artificial microRNA(amiRNA) via MultiSite Gateway protocols for thecreation of novel binary T-DNAvectors. Expression ofreporter genes in specific cell types illustrated thefunctionality of such vectors. Silencing of the endog-enous gene coding for phytoene desaturase withamiRNA in stable transgenic tomato plants demon-strated that such vectors can be used to efficientlycontrol transcript abundance in fruit tissues. In addi-tion, tomato driver lines were generated for the tran-scriptional activation of any responder transgenelocus, based on the LhG4 two-component system.Expression of several reporters and phenotypes in-duced within these lines facilitated their communaluse for genetic manipulations. The assembly of aSolanaceae genetic toolbox is an important asset forfuture functional analysis in tomato and related crops.

RESULTS AND DISCUSSION

Cloning Strategy and Structure of the Vector Collection

The Gateway MultiSite recombinational cloningframework is ideally suited for constructing versatilecollections of genetic elements to be assembled inpredetermined order, orientation, and open readingframe (ORF) registry (Karimi et al., 2005, 2007a, 2007b).The overall strategy underlying our vector resources isillustrated in Figure 1. Three types of elements havebeen considered so far: plant promoters, to direct thetranscription of any gene of interest in well-definedtomato tissues; ORFs, to produce the encoded proteins;and gene-specific sequence tags (GSTs) or amiRNAprecursors, to silence targeted genes.

Figure 1. Assemblage of differentgenetic elements captured into en-try clones via LR reaction to facil-itate genetic studies in tomato. B1,B2, B4, L1, L2, L4, and R1: corre-sponding att Gateway recombina-tion sites. Sp, spectinomycin; Sm,streptomycin; Km and Kan, kana-mycin bacterial and plant select-able markers, respectively; LB, leftT-DNA border; RB, right T-DNAborder; Prom, promoter; pdk-catintron, pHELLSGATE12-derived in-tron spacer; ccdB, counter-select-able bacterial marker; and 3#OCS,octopine synthase terminator.

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Each element was first captured in an entry clone,either via restriction/ligation cloning in a plasmidcarrying multiple restriction sites flanked by attL andattR recombination sites or via PCR amplification andaddition of flanking attB sites, followed by a BPClonase reaction. We chose to clone all promoters inattL4-promoter-attR1 cassettes and all other elementsin attL1-sequence-attL2 cassettes. The resulting entryclones can be recombined with various Gateway des-tination vectors in LR Clonase reactions to createexpression vectors. Interestingly, two or more ele-ments available as entry clones can be assembled viaa single MultiSite LR Clonase reaction in the order andorientation defined by the position of the attL and attRsites flanking the recombined sequences (Cheo et al.,2004; Karimi et al., 2007b). In our configuration, anypromoter can be positioned easily upstream of an ORF,a GST, or an amiRNA precursor and drive its tissue-specific transcription in transgenic plants. Lastly,novel modular destination vectors can be constructedfrom any expression clone via reverse BP Clonasereactions, further expanding the possible cloningschemes (Karimi et al., 2007b).

Fruit-Specific Promoters

Flowers and fruits are the main targets for geneticimprovement of fruit crops. However, these organs arethe last to form, and their manipulation with broadlyexpressed promoters is therefore restricted to non-pleiotropic genetic perturbations. Promoters activesolely in these organs bypass this limitation and per-mit the functional analysis of all genes involved infruit development. Such sequences facilitate the engi-neering of fruit quality, either directly or by providingproof-of-concept information from which strategiescan be developed to harness natural variation.As an initial set of regulatory sequences of interest,

we chose four promoters from tomato genes shownpreviously to be transcribed specifically at differentstages of fruit growth and maturation. In addition, weincluded a well-characterized Arabidopsis (Arabidop-sis thaliana) promoter of which the activity is restrictedto specific carpel and nectary tissues during flowerdevelopment (Table I). The five promoter sequencesare documented in Supplemental Table S1. The antic-ipated temporal and spatial transcriptional activity in

the fruit for each cloned promoter is summarizedbelow. The size of each captured sequence is alsoindicated in parentheses. They all end in a window of1 to 34 nucleotides upstream of their cognate ORF.

INHIBITOR OF MERISTEM ACTIVITY (0.5 kb)

INHIBITOR OF MERISTEM ACTIVITY (IMA; acces-sion number AM261628) encodes a 90-amino-acidtomato protein that harbors a putative central zincfinger domain similar to the recently characterizedMINI ZINC FINGER proteins (Hu and Ma, 2006;Sicard et al., 2008). While absent from vegetativeorgans, IMA expression is detected in all floral organsand to a greater extent in carpels and petals. However,the highest expression was observed in the developingfruit. IMA transcripts are detected as early as thepreanthesis stage corresponding to isolated carpels,accumulate gradually and strongly in fruit to reach amaximum at 10 d after anthesis (daa), and then de-crease to reach basal levels at the mature green stage.IMA acts as an inhibitor of cell proliferation duringflower termination (Sicard et al., 2008). Its promotersequence was identified via the Solanaceae GenomeNetwork (SGN) database in a BAC clone encompass-ing the IMA gene sequence (accession numberAC122544).

CRABS CLAW (3.7 kb)

The Arabidopsis CRABS CLAW (CRC) gene belongsto the YABBY transcription factor family (Bowman andSmyth, 1999). It is required for nectary development inArabidopsis flowers and for abaxial-adaxial polarityspecification of carpels. The CRC promoter is activethroughout carpel primordium initiation but becomesrestricted to the valve abaxial domain upon anthesis.In addition, it is expressed in central placental do-mains and in nectaries throughout their development(Lee et al., 2005). This promoter was chosen because itmarks the earliest stages of carpel initiation, a patternnot presently available from endogenous tomato pro-moters.

TPRP (2.6 kb)

TPRP is a cell wall tomato Pro-rich protein (Saltset al., 1991). TPRP expression levels are high in all

Table I. Genetic elements in entry clones

Class Element(s) Recipient pDONR Entry Clone att Sites

Promoter PPC2 pDONR P4-P1R pEN-L4-PPC2-R1 attL4–attR1Promoter TPRP pDONR P4-P1R pEN-L4-TPRP-R1 attL4–attR1Promoter IMA pDONR P4-P1R pEN-L4-IMA-R1 attL4–attR1Promoter CRC pEN-L4-R1 pEN-L4-CRC-R1 attL4–attR1Promoter PG pEN-L4-R1 pEN-L4-PG-R1 attL4–attR1Reporter enzyme SI pDONR221 pEN-L1-SI-L2 attL1–attL2amiRNA PDS pDONR221 pEN-L1-miRpds-L2 attL1–attL2Two component LhG4AtO pDONR221 pEN-L1-LhG4ATO4-L2 attL1–attL2

Flexible Tools for Gene Expression and Silencing in Tomato

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tissues of young tomato fruits, corresponding withcell division phases II and III of fruit development(Gillaspy et al., 1993). Promoter activity is dramaticallyreduced in mature green and ripe fruits and com-pletely absent in all other parts of the plant (Salts et al.,1991; Carmi et al., 2003). The TPRP promoter has beenused successfully to down-regulate the expression ofthe DE-ETIOLATED1 gene, resulting in an increase ofthe carotenoid and flavonoid content restricted to thefruit (Davuluri et al., 2005).

PPC2 (2.0 kb)

The PPC2 gene codes for a phosphoenolpyruvatecarboxylase isoform present in developing tomatofruits that is presumably involved in organic acidaccumulation and CO2 fixation. LYCes;PPC2 is highlyand specifically expressed during the phase of rapidfruit growth, corresponding to cell expansion. Expres-sion increases initially at the end of cell division anddecreases at the beginning of ripening. In younggrowing fruits, LYCes;PPC2 mRNA has been specifi-cally located by in situ hybridization in the pericarpand the gel surrounding the seeds (Guillet et al., 2002).Accordingly, the 2.0-kb promoter fragment has re-cently been shown to direct fruit-specific GUS expres-sion during the cell expansion phase in the placenta,gel, and later in the pericarp tissues in transgenictomato plants (C. Guillet and C. Rothan, unpublisheddata).

Polygalacturonase (4.8 kb)

Polygalacturonase (PG) is a cell wall hydrolaseabundantly secreted during fruit ripening and con-

tributing to fruit softening. PG expression is tightlyrelated to the ripening process. Transcriptional activa-tion at the onset of ripening results in a high level ofPG mRNA (Biggs and Handa, 1989; DellaPenna et al.,1989). Within the ripening fruit, a 1.4-kb promoterfragment has been shown to direct GUS expressionmostly to the outer region of the pericarp and the col-umella tissue in transgenic tomato plants (Montgomeryet al., 1993). However, a longer (4.8 kb) PG promoter waspreferred because it yields higher levels of chloram-phenicol acetyl transferase reporter activity in ripeningtransgenic tomato fruits (Nicholass et al., 1995) and hasbeen successfully used to engineer metabolite fruitcontent (Davidovich-Rikanati et al., 2007; Kovacs et al.,2007).

The PPC2, TPRP, and IMA promoters were ampli-fied by PCR with sense and antisense primers includ-ing the attB4 and attB1 sequences, respectively. Theamplified fragments were recombined with pDONRP4-R1 to produce the corresponding entry clones. Thelarger PG and CRC promoters were introduced byconventional restriction/ligation cloning into pEN-L4-R1 in which a multicloning site is flanked by the attL4and attR1 sequences.

Expression of the GUS Reporter in Tomato Fruits

In the context of MultiSite Gateway expressionconstructs, the described promoters (pEN-L4-promoter-R1) were fused to theGUS gene (pEN-L1-SI-L2; Karimiet al., 2007b) in the pK7m24GW destination vector viaMultiSite LR Clonase reaction (Karimi et al., 2005;Table II). Initial validation of promoter activity wasachieved with a previously described fruit transientexpression assay (Orzaez et al., 2006). Briefly, the

Table II. Expression vectors

Destination and Modular

VectorRecombined Entry Clones

Resulting Expression

Vector

pK7m24GW pEN-L4-PPC2-R1 3 pEN-L1-SI-L2 pXK7SIPPC2pK7m24GW pEN-L4-PG-R1 3 pEN-L1-SI-L2 pXK7SIPGpK7m24GW pEN-L4-TPRP-R1 3 pEN-L1-SI-L2 pXK7SITPRPpK7m24GW pEN-L4-IMA-R1 3 pEN-L1-SI-L2 pXK7SIIMApK7m24GW pEN-L4-CRC-R1 3 pEN-L1-SI-L2 pXK7SICRCpMK7S*NFm14GW pEN-L4-PPC2-R1 pXK7S*NFPPC2pMK7S*NFm14GW pEN-L4-PG-R1 pXK7S*NFPGpMK7S*NFm14GW pEN-L4-TPRP-R1 pXK7S*NFTPRPpMK7S*NFm14GW pEN-L1-IMA-R1 pXK7S*NFIMApMK7S*NFm14GW pEN-L4-CRC-R1 pXK7S*NFCRCpK7GW2 pEN-L4-miRpds-L2 pXK7miRpds2pK7m24GW pEN-L4-PPC2-R1 3 pEN-L1-miRpds-L2 pXK7miRpdsPPC2pK7m24GW pEN-L4-PG-R1 3 pEN-L1-miRpds-L2 pXK7miRpdsPGpK7m24GW pEN-L4-TPRP-R1 3 pEN-L1-miRpds-L2 pXK7miRpdsTPRPpK7m24GW pEN-L4-IMA-R1 3 pEN-L1-miRpds-L2 pXK7miRpdsIMApK7m24GW pEN-L4-CRC-R1 3 pEN-L1-miRpds-L2 pXK7miRpdsCRCpK7m24GW pEN-L4-PPC2-R1 3 pEN-L1-LhGATO4-L2 pXK7LhGATO4PPC2pK7m24GW pEN-L4-PG-R1 3 pEN-L1-LhGATO4-L2 pXK7LhGATO4PGpK7m24GW pEN-L4-TPRP-R1 3 pEN-L1-LhGATO4-L2 pXK7LhGATO4TPRPpK7m24GW pEN-L4-IMA-R1 3 pEN-L1-LhGATO4-L2 pXK7LhGATO4IMApK7m24GW pEN-L4-CRC-R1 3 pEN-L1-LhGATO4-L2 pXK7LhGATO4CRC

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promoter-GUS binary expression vectors were trans-formed intoAgrobacterium tumefaciens and injected intofruits of different developmental stages, from 20 daa tobreaker + 11 d (Br+11). Several days after injection,fruits were sampled and thin sections were analyzedfor GUS activity via classical histochemical assays(Supplemental Fig. S1 and Supplemental Materialsand Methods S1). GUS staining consistent with previ-ously published expression results were obtained forlate stage promoters, such as in PGpro:GUS-injectedripening fruits, but the assessment of the promoteractivity in the transient assay was restricted to morethan 20-daa fruits and concurrent with wounding andstress. These technical constraints probably explainwhy temporal and spatial expression might be partlyartifactual in agroinjected fruits. For example, theearly-stage IMA promoter was active in ripeningfruits, and higher level of GUS staining was observedin placenta and gel, presumably because of a betterpenetration of Agrobacterium in these tissues. Never-theless, transient expression data provided the initialevidence that the Gateway cloning framework wascompatible with gene expression in tomato, a prereq-

uisite for in-depth analysis of promoter activity instable transgenic plants.

To this end, the MicroTom cultivar was agroinfectedwith an improved protocol yielding transformationfrequency consistently .80% (see “Materials andMethods”). The ploidy of regenerated plantlets wasmeasured with flow cytometry, and polyploid plantswere discarded. Between 12 and 25 independentlytransformed plants were generated for each promoter-GUS transgene (Table II). Following GUS staining andselection of plants with one or two transgene copies,two representative lines were selected for each con-struct, and the GUS staining pattern of 10 kanamycin-resistant T1 plants per original line was characterizedat specific stages of development: flower, 6-mm buds(approximately stage 9 according to Baldet et al., 2006),and anthesis; fruit, 6 daa (cell division), 10 daa, 15 daa(cell expansion stage; Lemaire-Chamley et al., 2005),mature green (MG; transition to ripening), and Br+7(ripe fruit). GUS staining of T1 leaves and entire youngT2 plantlets completed the analysis.

As expected, strong GUS activity was observed in all35Spro-GUS tissues, while no staining was detected in

Figure 2. GUS activity in tomato (cv MicroTom) stably transformed with promoter:GUS transgenes generated via MultiSiteGateway cloning. Bars = 10 mm.





wild-type controls (Fig. 2). The five selected fruitpromoters only resulted in fruit-specific expression,with no GUS staining in vegetative organs (Fig. 2).Flower buds showed weak GUS staining only in theIMApro:GUS, TPRPpro:GUS, and PPC2pro:GUS lines.

In agreement with earlier reports (Sicard et al.,2008), IMApro:GUS fruits displayed weak but consis-tent GUS staining at early stages of development,particularly in placental and vascular tissues. Addi-tional staining of seed tegument was observed in ripefruit (Fig. 2). In CRCpro:GUS fruits, GUS staining wasrestricted to the outer carpel wall from early stagesuntil MG (Fig. 2). This result is reminiscent of the CRCnative expression that is largely restricted to the ab-axial epidermis in the Arabidopsis carpel (from thebase to the tip of the carpel, along its entire circum-ference), where it promotes abaxial cell fate (Eshedet al., 1999). In TPRPpro:GUS fruits, GUS stainingpeaked at the immature green stage (Fig. 2), consistentwith earlier reports (Gillaspy et al., 1993). In PPC2pro:GUS fruits, strong GUS staining was limited to thefruit expansion phase (Fig. 2), in agreement withprevious data (Guillet et al., 2002; C. Guillet and C.Rothan, unpublished data). In PGpro:GUS fruits, GUSactivity was not detected in early stages, was weakat the onset of ripening (MG), and high in the ripefruit (Br+7; Fig. 2), again as published previously(Montgomery et al., 1993).

In summary, the temporal regulation of the fivepromoters observed in stable transgenic tomato fruitswas in agreement with the data reported in wild-typeor stable transgenic plants. In addition to markingselected stages of fruit development, several testedpromoters also offer the possibility to direct transgeneexpression to specific fruit tissues. For example, theCRC promoter can specifically target genes expressedin the epidermis of developing tomato fruit and in-volved in cuticle or phenylpropanoid synthesis(Mintz-Oron et al., 2008).

GFP Markers Specific to Fruit Cell Types

Promoter-GUS transgenes were useful to reporttissue-specific transcription. Yet higher spatial resolu-tion can be achieved via the transcriptionally regu-lated expression of a nuclear localization signal (NLS)fused to fluorescent proteins. Such markers enable theselection of particular cell types, as well as the trackingof cell proliferation and enlargement in the course offruit development. Therefore, we generated a promoter:NLS-GFP-GUS construct for each of the five selectedpromoters via LR recombination into the modulardestination vector pK7S*NFm14GW (Karimi et al.,2007b; Table II). Stable tomato (cv MicroTom) reporterlines carrying one of each promoter:NLS-GFP-GUStransgene (Table II) were generated, screened, andanalyzed as described above. In addition, GFP pro-duction was verified in fresh 150-mM-thick pericarpsections with epifluorescence microscopy. Promoter:

GUS and promoter:NLS-GFP-GUS lines displayed sim-ilar GUS staining patterns (data not shown).

The NLS fused to GFP proved very effective becausethe fluorescence signal was detected in the nucleus ofall cell types in the pericarp and gel of 35Spro:NLS-GFP-GUS fruits (Fig. 3, A and B). In the pericarp ofPPC2pro:NLS-GFP-GUS fruits, GFP was absent fromthe fruit epidermis and from most underlying divid-ing cells and was essentially limited to the largeexpanding mesocarpic cells (Fig. 3, C and D). Thisobservation is consistent with the hypothesis thatmalate synthesized via PECase serves as a counterionfor potassium that accumulates in the vacuole, thusproviding the driving force for fruit cell enlargement(Guillet et al., 2002).

Figure 3. Detection of nuclear GFP in cells of tomato fruit pericarp.Sections were prepared from 8-daa fruits. A and B, 35Spro:NLS:GUS-GFP tissues showing GFP signal in all fruit tissues. B, Magnified imageshowing localization of GFP in the nucleus. C and D, Fruit pericarpcross section from 8-daa tomato fruits transformed with PPC2pro:NLS:GUS-GFP showing preferential GFP localization in expanding cellsfrom fruit mesocarp. D, Magnified image of mesocarp showing local-ization of GFP in polyploid nucleus from large cell. Bars = 400 mm in Aand C and 100 mm in B and D.

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To conclude, in addition to the information pro-vided by promoter-GUS transgenic lines about expres-sion targeted at specific developmental stages and inparticular tissues, promoter:NLS-GFP-GUS lines can beused to define the spatial distribution of a transgenedriven by a selected promoter in tomato fruits withhigher cellular resolution.

Vectors for Silencing Genes in Fruits

RNA-mediated gene silencing, or RNA interference,is a method of choice to generate loss-of-functionphenotypes caused by partial to complete gene knock-down. amiRNAs have been shown to efficiently si-lence genes inmultiple plant species, including tomato(Alvarez et al., 2006; Schwab et al., 2006; for review, seeOssowski et al., 2008). This approach entails the ex-pression of an endogenous plant microRNA precursorengineered to yield a 21-nucleotide double-strandedamiRNA that is chosen to silence a gene, or severalgenes, of interest according to experimentally definedtarget selection parameters. The DNA sequence cod-ing for an amiRNA can easily be synthesized byoverlapping PCR amplification (Schwab et al., 2006)with the addition of the attB1 and attB2 recombinationsites at their 5# and 3# ends, respectively, and subse-quently captured as an amiRNA entry clone (attL1-amiRNA-attL2). Once in this format, any amiRNAprecursor can be cloned downstream of any promoterof interest, including those described above, in asimple MultiSite LR Clonase reaction. The additionof attB-flanking sites to an amiRNA precursor (i.e. tothe MIR319a backbone in this particular case) did notalter its ability to silence a target gene in Arabidopsis(Supplemental Fig. S2 and Supplemental Materialsand Methods S1).

To confirm silencing in tomato, we generated Micro-Tom lines in which the PDS gene was silenced via thetranscription of a matching amiR-pds precursor underthe control of the cauliflower mosaic virus (CaMV) 35Sor PPC2 promoter (Tables I and II). The PDS enzyme isinvolved in carotenoid biosynthesis and protects chlo-rophyll from photooxidation in developing tissues.PDS silencing causes photobleaching that can be read-ily recorded in the course of development. Bleachingwas observed in all tissues of the 35Spro:amiR-pdstomato plants (Fig. 4, B and C). The most severely af-fected plantlets did not survive in the greenhouse (datanot shown). In contrast, the 18 PPC2pro:amiR-pdsprimary transformants did not display any visualphenotype during vegetative growth. However, one-third of these T0 plants produced fruits that remainedlight orange during ripening and only turned light redwhen left on the plant (Fig. 4, D and E). Our observa-tions are consistent with the PPC2 profile documentedthrough reporter expression and indicating that thepromoter activity is the highest at the early stages offruit development (Fig. 2) and in the mesocarp (Fig.3C), i.e. at developmental stages and in fruit tissuesless sensitive to light than the exocarp where the 35S-driven expression is very high (Fig. 3A) and thephotobleaching more likely to occur. Our results fur-ther illustrate how fruit promoters and constructsdeveloped herein can be used to target specific fruittissues and alter expression of candidate genes intomato.

Because amiRNA target recognition relies on a short21-nucleotide sequence, amiRNAs yield more specificsilencing than the numerous small interfering RNAsderived from the long (hundreds of base pairs) double-stranded hpRNA molecules. However, because manygenes encoded in Solanaceae have yet to be identified,

Figure 4. Tissue-specific amiRNA silenc-ing in tomato fruits. Flowers at anthesis(top), mature leaves (middle), and ripefruits (Breaker +7; bottom). A, The wildtype (cv MicroTom). B and C, 35Spro:amiR-pds plants with strong to extremephotobleaching in all organs. D and E,PPC2pro:amiR-pds plants with moderateand transient photobleaching restricted tothe fruit. Bars = 10 mm.





hpRNA silencing is still valuable: for example, toavoid the degradation of a gene different from thetarget but serendipitously containing sequencesmatch-ing a selected amiRNA or to silence simultaneouslymultiple closely homologous transcripts.

Therefore, we constructed a new series of hpRNAexpression vectors with the aim to restrict gene silenc-ing to specific tissues or organs (Supplemental TableS5). For this purpose, we implemented novel cloningschemes involving only BP and LR Clonase reactionsto position any promoter available as an entry clonein front of a silencing transgene (Supplemental Figs.S3–S5 and Supplemental Materials and Methods S1).The constructs generated were derived from thepHELLSGATE12 vector (Wesley et al., 2001; Helliwelland Waterhouse, 2003; Hilson et al., 2004) in which thesequence targeted for silencing (GST), originallycaptured in an entry clone (attL1-GST-attL2), wastransferred simultaneously in two independent Gate-way cassettes separated by an intron spacer (attR1-ccdB-attR2-intron_spacer-attR2-ccdB-attR1). This spacerconsisted of two head-to-head introns that enabledsplicing of the encoded transcript regardless of itsorientation. In this configuration, the attR1 and attR2recombination sites were inverted with respect to oneanother, so that the two copies of the target sequencewere inserted as inverted repeats into the resultinghpRNA expression clone (Helliwell and Waterhouse,2003).

For both hpRNA and amiRNA silencing, software isavailable that selects the most likely specific targetsites taking into consideration entire genome se-quences: for example, SPADS for the design of GSTscloned into hpRNA expression vectors (Thareau et al.,2003; Sclep et al., 2007) and Web MicroRNA Designerfor the selection of amiRNA sequences introduced intothe microRNA precursor backbone by overlappingPCR (http://wmd3.weigelworld.org/). Nevertheless,some amiRNAs and hpRNAs fail to yield efficientsilencing, and current algorithms cannot accuratelypredict silencing efficiency.

Driver Constructs and Tomato Lines

In tomato, as in most crop plants, transformationprotocols require tissue culture. However, becausemany tissue- and cell-type-specific promoters displayextensive expression in callus, transgenes with dele-terious effects might be selected against during tissueculture. A solution to this problem is to dissociate thetarget gene expression locus from its transcriptionalregulator. For this purpose, we established referencetomato driver lines facilitating both gene misexpres-sion and silencing. These lines were constructed withelements of the pOp/LhG4 system engineered fortranscriptional activation into plant species (Mooreet al., 1998; Rutherford et al., 2005; Wielopolska et al.,2005; Ori et al., 2007). Its main components are theLhG4 chimeric transcription factor, consisting of thebacterial LacI DNA-binding and yeast GAL4 activa-

tion domains, and the synthetic pOp promoters con-taining multiple copies of the lac operator bound byLhG4. The driver locus codes for the LhG4 gene underthe control of a promoter with a particular spatio-temporal pattern. The responder locus contains asynthetic pOp promoter driving the transcription ofa gene, hpRNA, or amiRNA of interest and is onlyactive in the presence of LhG4. Both loci were com-bined in the same plant either by crossing or bytransformation.

Figure 5. Tissue-specific transactivation in tomato with selected Arab-idopsis promoters. A and B, Expression of dsRED or GUS, activated intrans by the organ primordia and abaxial domain pFIL:LhG4 driver.Abaxial expression is evident after leaf primordium has expanded. Cand D, Flower-specific growth arrest stimulated by specific transacti-vation of a pOP:KAN1 reporter, with the pAP1:LhG4 driver. E and F,Petal-only radialization stimulated by specific transactivation of a pOP:KAN1 reporter, with the pAP3:LhG4 driver. G and H, Carpel-specificgrowth arrest stimulated by transactivation of the pOP:KAN1 reporterwith the pCRC:LhG4 driver. WT, Wild type; SlKAN, KANADI1 gene oftomato.

Fernandez et al.




First, we created an array of reporter lines to vali-date the use of the pOp/LhG4 system in tomato,including pOP:GUS, pOP:ER-GFP, pOP:dsRFP, andpOP:KANADI1 (KAN1). The first three reporters wereuseful for the detailed examination of promoter activ-ity. The pOP:KAN1 reporter drove the potent tomatoKANADI1 gene that imposes abaxial identity andsevere growth arrest whenever expressed ectopicallyoutside of its normal domain (Eshed et al., 2001). Thisreporter provided a simple and sensitive test forspatial activity of any examined driver line becauseeven low level of expression, possibly escaping detec-tion with the other reporters, might result in clearlyvisible phenotypes. The KAN1 reporter is particularlyrelevant to reveal background activity within largeorgans, such as the tomato fruits, that are difficult tocharacterize via classical histological staining or mi-croscopy analysis.Second, we crossed these reporter lines with tomato

driver lines in which the LhG4 transgene was underthe control of one of five Arabidopsis flower-specificpromoters (described in Pekker et al., 2005). Signifi-cantly, the Arabidopsis FIL, AP1, AP3, and CRC pro-moters maintained their specific expression domainsin F1 tomato plants expressing both a driver andreporter construct. The FIL promoter stimulated ex-pression in all organ primordia but not in the shootapical meristem proper (Fig. 5, A and B). The AP1promoter drove expression in the sepals, petals, andflower meristems because all were strongly inhibitedupon transactivation of KAN1 (Fig. 5, C and D) and theAP3 promoter only in the petals and stamens, of whichthe petals became radialized upon transactivation ofKAN1 (Fig. 5, E and F). The CRC promoter stronglyinhibited carpel development upon KAN1 transacti-vation but nowhere else (Fig. 5, G and H). Only onepromoter, FUL, failed to show the expected earlyexpression in the pericarp (data not shown). Theproportion of observed versus expected patterns wasin the same range as that obtained when attempting tosystematically recapitulate transcription pattern withcloned promoter sequences (Lee et al., 2006). To con-clude, defined Arabidopsis promoters are a usefulresource to misexpress or silence genes in other spe-cies, such as tomato.Finally, we have expanded the repertoire of driver

constructs by generating via MultiSite LR recombina-tion all of the transcriptional fusions between each ofthe tomato fruit promoters described above and theLhG4AtO ORF, optimized for expression in plants(pEN-L1-LhATOG4-L2; Karimi et al., 2007b; Table II).

Perspectives

All plasmids with tomato sequences and tomatoseed stocks described here are made available via theNottinghamArabidopsis Stock Centre (NASC; http://Arabidopsis.info) to ensure long-term distribution andappropriate documentation of the materials. The cen-tralization of resources and related information at the

SGNWeb site (Mueller et al., 2005) and complementedat NASC will also facilitate their integration intoreference genome browsers as well as genotype andphenotype databases. As demonstrated by the Arabi-dopsis community, shared genetic tools promote rapidand significant scientific achievements, which is par-ticularly relevant for a plant family of high commercialvalue. For example, the tomato promoters available asGateway entry clones might be used to characterizegenes involved in key processes taking place duringthe successive phases of fruit development, namely,cell proliferation (TPRP and CRC; mitotic cycle), cellexpansion (PPC2; endoreduplication, starch synthesisand storage, and cell wall synthesis), and ripening(PG; cell wall and starch breakdown and soluble sugarand carotenoid accumulation), or in specific fruit tis-sues (IMA, vascular tissues; CRC, cuticle formationand secondary metabolism). Promoters only, ormainly, active in flower or fruit should avoid some ofthe common pitfalls associated with strong constitu-tive promoters (such as CaMV 35S) that also affectvegetative plant parts and therefore hamper the anal-ysis of gene function, as clearly illustrated for PDSamiRNA silencing in tomato.

Finally, it is worth noting that the Gateway clonesdescribed here and in a recent companion article(Estornell et al., 2009) are compatible with a widerange of complementary entry clones (coding foralternative terminators, reporters, or activators) anddestination vectors designed for various functionalassays in plants or in other heterologous systems (forreview, see Karimi et al., 2007a).

MATERIALS AND METHODS

Bacterial Strains

Host Escherichia coli strains were either DH5a (with entry and expression

clones) or DB3.1 (with destination vectors; Invitrogen). Bacterial cultures were

grown at 37�C in Luria broth medium with appropriate antibiotics.

BP and LR Clonase Reactions

Additional information about the basic Gateway site-specific recombina-

tional cloning protocols is provided online by the manufacturer (http://www.

invitrogen.com/). Entry clones were created in BP Clonase reactions with 50

to 100 ng of PCR product and 50 ng of donor vector. Expression clones were

created in LR Clonase II reactions with 30 ng of each entry clones and 50 to 80

ng of destination vector. Reactions were done in 10 mL total volume containing

2 mL of BP or LR Clonase, incubated at 25�C overnight. The reaction was

inactivated by addition of 1 mL of proteinase K, and 5 mL from each reaction

was used to transform E. coli competent cells as described.

Promoter Entry Clones

Specific oligonucleotides were designed to amplify the PPC2, TPRP, and

IMA promoters, including the attB4 and attB1 sites upstream of the 5# and 3#gene-specific priming sequences, respectively (Supplemental Table S2). Pro-

moters were amplified by PCR with either tomato genomic or plasmid DNA

as template. PCR mix contained 0.5 mM of each primer, 25 ng template DNA,

200 mM of each dinucleotide triphosphate, 2.5 units of Platinum HiFi DNA

polymerase (Invitrogen), 13 PCR buffer, and 1 mM MgSO4. Programmed

cycles were as follows: 5 min initial denaturing step at 94�C; 30 cycles of 30 s

denaturation at 94�C, 30 s annealing at 50�C, 1 to 3 min extension (1 min per kb)





at 68�C, and 5 min termination at 68�C. PCR products were purified with the

High Pure PCR Purification Kit (Roche Diagnostics). BP Clonase recombination

reactions were done with the purified PCR products and pDONR P4-P1R to

generate the entry clones. After transformation into DH5a competent cells,

colonies were screened by PCR for the correct insert size and clones were

sequence validated.

Large DNA fragments are more difficult to capture into entry clones.

Because the PG and CRC promoters were 4.8 and 3.7 kb, respectively, we chose

conventional restriction/ligation techniques to build the corresponding entry

clones. The PG promoter originally cloned into pBIN19 (Nicholass et al., 1995)

was isolated as a fragment obtained by BamHI and partial HindIII digestion.

The isolated fragment was ligated to pBlueScript (CLONTECH) plasmid

linearized by BamHI-HindIII digestion to generate an intermediate clone. Two

complementary PG fragments were ligated at once into SalI-XhoI-linearized

pEN-L4-R1: from 5# to 3#, a XhoI-XbaI fragment isolated from the PG

intermediate clone, and an XbaI-XhoI fragment isolated from the original PG

pBIN19 derivative. The plasmid pEN-L4-R1 carried a multiple cloning site

flanked by the attL4 and attR1 recombination sites (attL4-XmnI-SalI-BamHI-

KpnI-ccdB-XhoI-attR1; O. Nyabi, M. Karimi, P. Hilson, and J. Haigh, unpub-

lished data). The CRC promoter originally cloned into pBS (Lee et al., 2005)

was isolated as a SalI-XhoI fragment and cloned into the same sites within

pEN-L4-R1. Digested fragments were separated by electrophoresis in agarose

gels, extracted, and purified with the High Pure PCR Purification Kit (Roche

Diagnostics). After transformation into DH5a competent cells, clones were

screened by restriction analysis to identify plasmids with the expected insert

in the correct orientation and subsequently sequence validated.

Creation of amiRNAVectors

According to Schwab et al. (2006), the plasmid pRS300 was used as

template to introduce an amiRNA sequence targeting the tomato (Solanum

lycopersicum) and Arabidopsis (Arabidopsis thaliana) PDS gene (5#-TCCATG-

CAGCTACCTTTCCAC-3#) into the miR319a precursor by site-directed muta-

genesis. Overlapping PCR amplification steps were done as described in the

original cloning protocol except that, in the final PCR reaction, the oligonu-

cleotides A and B (based on the pBSK backbone) were replaced by two

alternative oligonucleotides, attB1-amiRNA-fw and attB2-amiRNA-rev, car-

rying the attB1 and attB2 sites, respectively. These two primers can be used for

the amplification of any amiRNA derived from the miR319a precursor

backbone (see Supplemental Materials and Methods S1 for details). The

resulting PCR product containing the amiR-pds precursor was cloned into

pDONR221 by BP Clonase reaction, resulting in the pEN-L1-miRpds-L2 entry

clone (Table I) and then subcloned into the pK7WG2 or pK7m24GW destina-

tion vectors by LR Clonase reaction to yield the expression vectors driving

amiR-pds silencing under the control of the CaMV 35S, PPC2, PG, TPRP, IMA,

or CRC promoters (Table II).

Transactivation Clones

Reporter OP clones were sequence verified after restriction-ligation sub-

cloning downstream of an OP array (10xOP-TATA-MCS-3#OCS in BJ36) and

transferred into the NotI site of the pART27 binary vector. pOP:GUS and pOP:

RFP were described previously (Lifschitz et al., 2006). pOP:KAN1 was gener-

ated by PCR amplification of the ORF of the tomato KAN1 ortholog, desig-

nated SGN-U321055. Driver lines were generated by transcriptional fusion of

promoters in front of the chimeric LhG4 (MCS-LhG4-3#OCS in BJ36; Moore

et al., 1998) and transferred into the NotI site of pART27. The Arabidopsis

LhG4 promoter lines were described before (Pekker et al., 2005), but briefly, a

6.1-kb FILAMENTOUS FLOWER, a 1.7-kb APETALA1, a 0.5-kb APETALA3,

and 3.5-kb CRC Arabidopsis promoter fragments were subcloned in front of

LhG4 in pART27.

Tomato Transformation and Transactivation

MicroTom tomato lines were transformed with the Agrobacterium tumefa-

ciens strain GV3101. Briefly, 8-d-old MicroTom cotyledons were cut at both

extremities and in the middle, placed on solid KCMS in petri dishes,

incubated at 25�C for 24 h, soaked for 30 min with shaking in Agrobacterium

suspension (Agrobacterium was grown in Luria broth to OD = 1, pelleted, and

resuspended in liquid KCMS to OD = 0.05–0.08), blotted dry on sterile

Whatman paper, and incubated on solid KCMS in petri dishes in the dark for

48 h at 25�C. For regeneration, the cotyledons were laid on 2Z medium with

250 mg L21 timentin (ticarcillin and clavulanate; GlaxoSmithKline) and 100

mg L21 kanamycin and incubated during 15 d at 25�C in the light. Regener-

ation medium was changed every 15 d when necessary, with timentin

concentration reduced to 150 mg L21. In case of Agrobacterium overgrowth,

timentin concentration was kept at 250 mg L21. Regenerated plantlets were

transferred to rootingmedium supplementedwith 100mg L21 kanamycin and

75 mg L21 timentin. With this protocol and the vectors described herein,

transformation frequencies observed using MicroTom cotyledons were con-

sistently .80% and up to 90%. Less than 15% of the transgenic plantlets were

polyploid according to flow cytometry analysis. Cultivation from cotyledon

explants to transfer of transgenic plants into the greenhouse lasted 2 to 3

months, and 2 to 3 additional months were required to collect transgenic

seeds. The composition of in vitro culture media is provided in Supplemental

Tables S3 and S4.

Cotyledon transformation of M82 tomato and leaf discs were carried out

according to McCormick (1991) with the Agrobacterium strain GV3101.

promoter:LhG4 lines were crossed to 10xe lines to generate the F1 promoter..reporter plants.

Upon request, all novel materials described will be made available in a

timely manner for noncommercial research purposes, subject to the requisite

permission from any third-party owners of all or parts of the material.

Obtaining any permissions will be the responsibility of the requestor.

GUS Histochemical Staining

Tissues from stably transformed tomato plants were soaked, vacuum-

infiltrated, and incubated for 30 min (PG), 1 h (35S, PPC2, and TPRP), 2 h

(CRC), or overnight (IMA) at 37�C in GUS staining solution [0.5 mM X-gluc,

0.15 M NaH2PO4, pH 7, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6, and 0.05% Triton

X-100]. GUS-stained tissues were cleared overnight in 100% ethanol and

stored in 70% ethanol.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. GUS activity in tomato fruits (cv MicroTom)

agroinjected with promoter:GUS transgenes generated via MultiSite

Gateway cloning.

Supplemental Figure S2. Albino phenotype resulting from PDS silencing

in Arabidopsis.

Supplemental Figure S3. Creation of novel hpRNA destination vectors.

Supplemental Figure S4. Construction of the intron-spacer donor vector

pDONR P1-R2-I-R2-P1.

Supplemental Figure S5. Alternative scheme to generate specific hpRNA

destination vectors.

Supplemental Table S1. Theoretical promoter sequences.

Supplemental Table S2. Primers designed for PCR amplification of genetic

elements and for validation of recombined plasmids.

Supplemental Table S3. Composition of cv MicroTom transformation

media (per liter).

Supplemental Table S4. Vitamins for cv MicroTom transformation media.

Supplemental Table S5. Specific hpRNA destination vectors.

Supplemental Materials and Methods S1.

ACKNOWLEDGMENTS

We thank Wilson Ardilez for sequencing and Martine De Cock for help in

preparing the manuscript.

Received September 16, 2009; accepted September 30, 2009; published October

7, 2009.

Fernandez et al.




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